Typically, urban wildlife communities are made up of generalist species that are adept at utilizing human resources. However, many wildlife species struggle in the face of extensive urbanization and would benefit from increased conservation of urban green space, increased urban landscape connectivity, and proactive wildlife population management strategies. Unfortunately, maintaining and/or increasing the availability of quality habitat for biodiversity conservation in urban areas can be challenging as these conservation efforts are often influenced by the decreasing availability of critical resources and the challenges in allocating those resources among competing socioeconomic and environmental needs. Therefore, to improve the management and conservation of urban wildlife, accurate measurements of potential trade-offs between the environmental, economic, and social goals and management actions of a city’s sustainable development plan are needed. Until now, much of the effort in wildlife habitat modeling and biodiversity mapping has been across large geographic areas or broad spatial scales. Those efforts have provided valuable insights into overall biodiversity patterns, identifying key hotspots, and understanding large-scale ecological processes. However, in urban environments, the dynamics of wildlife, habitat availability, and ecosystem services operate differently than in natural or rural landscapes. As urbanization continues to expand, there is a growing need to focus on fine-scale factors to address specific conservation challenges in urban systems. This research seeks to address some of these challenges and demonstrates how new and traditional species-relevant geospatial datasets can be leveraged in urban planning and design to drive local-scale conservation decisions that put biodiversity in the forefront. This work links long-term, multi-taxon, wildlife survey data and high-resolution land use and land cover datasets (1m) to determine where high-quality, well-connected habitats exist, or could most easily be justified and acquired, within the District of Columbia. This work also evaluates the spatial patterns of ecosystem service provisions across the urban landscape to identify “win-win” areas for conservation or restoration that will benefit both biodiversity and human wellbeing. Finally, the work evaluates a local translocation effort of the vulnerable eastern box turtle (Terrapene carolina carolina) to inform mitigation strategies when a sudden loss of habitat in an urban environment is inevitable. This research is particularly relevant to wildlife managers and urban planners in highly urbanized areas, where large parcels of land with suitable habitat are minimal and municipal environmental departments are often under-resourced. Local policymakers interested in incentivizing conservation efforts to meet state or national goals can use this information for strategic urban conservation initiatives.